Plasma concentrate and tissue sealant methods and apparatuses for making concentrated plasma and/or tissue sealant

ABSTRACT

An inexpensive device with a disposable cartridge for preparing tissue sealant is disclosed. The device is particularly applicable to star preparation of autologous tissue sealant. A method of sealing tissue in which the tissue sealant is applied immediately after mixing platelet-rich plasma concentrate (from the device) with a solution of calcium and thrombin is also disclosed. Preparation in the operating room of 5 cc sealant from 50 cc patient blood requires less than 15 minutes and only one simple operator step. There is no risk of tracking error because processing can be done in the operating room. Chemicals added may be limited to anticoagulant (e.g., citrate) and calcium chloride. The disposable cartridge may fit in the palm of the hand and is hermetically sealed to eliminate possible exposure to patient blood and ensure sterility. Adhesive and tensile strengths are comparable or superior to pooled blood fibrin sealants made with precipitation methods. Antifibrinolytic agents (such as aprotinin) are not necessary because the tissue sealant contains high concentrations of natural inhibitors of fibrinolysis from the patient&#39;s blood. The tissue sealant also contains patient platelets and additional factors not present in available fibrin sealants that promote wound healing.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention concerns compositions, methods and apparatuses formaking concentrated plasma and tissue sealant, for sealing tissue, forrapid separation of higher molecular weight components in mixtures, forrapid separation of blood plasma and for rapid separation of particulatecomponents in mixtures. The present invention is particularly applicableto preparation and use of autologous tissue sealant.

2. Discussion of the Background

Fibrin Glues and Sealants

Various substances have been tried to meet the need for a suitabletissue adhesive for use in surgical procedures. Completely syntheticmaterials such as cyanoacrylate have been tried and been found wanting.Natural fibrin glues and sealants made from blood components wererecognized early in the development of this technology. Surgical "fibrinsealants" (sometimes called "fibrin glues") made up of human fibrinogenactivated by bovine thrombin are used extensively in Europe. Such fibrinsealants have been shown to be superior to synthetic adhesives ortraditional surgery in many situations. In addition they reduce the needfor blood transfusions. Academic and surgical opinion on fibrin sealantsis very favorable. A recent review says:

Fibrin sealants are the most successful tissue adhesives to date. Theyhave many advantages over adhesive technologies such as cyanoacrylatesand marine adhesives in terms of biocompatibility, biodegradation andhemostasis. There are several commercial products in Europe but none inthe United States due to the current regulatory stance against pooledplasma blood products (Sierra, 1993; vide infra).

In current practice, fibrin sealant is made by isolating a concentrateof human fibrinogen, fibronectin and factor XIII, usually bycryoprecipitation, and combining it immediately before use with bovine(or sometimes human) thrombin. The thrombin converts the fibrinogen tofibrin which rapidly gels to form a transient hemostatic plug, which isthen stabilized by factor XIII. Fibronectin provides adhesion for cellsrepopulating the clot and tissue. The most common method of applicationof fibrin sealant is mixing of concentrated fibrinogen from pooled humanblood with bovine thrombin and calcium immediately before use.

Fibrin sealant is not available in the U.S. commercially, but is inEurope (TISSEEL®, TISSUCOL®/Immuno; BERIPLAST®/Behring). Many papershave been published on its use.

Use of fibrin sealant in the United States is limited to preparationwithin the clinic outside FDA control. The reasons for FDA reluctance toapprove these products in the United States are:

virus transmission (e.g., HIV, hepatitis) from pooled human blood¹ and

immunological reactions to bovine thrombin², for example thrombin andfactor V inhibitors or foreign body reactions³.

These FDA concerns are so serious that the FDA has not approved anyfibrin sealant product, despite strong interest from surgeons and veryfavorable comparative studies in the literature.

These circumstances have led to much attention being given to methods toisolate an autologous counterpart of the fibrinogen containing componentin the TISSUCOL system in a practical manner. These efforts arediscussed in the reviews cited below. The following from a review byThompson⁴ shows that the value of star autologous fibrinogen is muchanticipated:

Fibrin glue is composed of two separate solutions of fibrinogen andthrombin. When mixed together, these agents mimic the last stages of theclotting cascade to form a fibrin clot. Fibrin glue is available inEurope but is not commercially available in the U.S.; therefore,investigators have extemporaneously compounded their own fibrin glue.Fibrinogen can be obtained from pooled, single-donor, and autologousblood donors and is usually isolated by the process ofcryoprecipitation. . . . The safest preparations use the patient's ownblood to prepare fibrin glue. . . . Use of. . . autologous blood resultsin minimal risk of disease transmission, but it requires anticipated useas in an elective procedure. The use of autologous blood usually is notpossible in trauma or an emergency surgical procedure.

Siedentop⁵ describes a number of approaches to the precipitation offibrinogen from plasma in the context of the proposed use of thismaterial as the fibrinogen furnishing component of a fibrin glue. Fourmethods were suggested: precipitation with ethanol, use ofunfractionated plasma, cryoprecipitation, and precipitation withammonium sulfate. Epstein⁶ suggests the use of a fibrinogen preparationfrom autologous plasma obtained using polyethylene glycol precipitation.A system for preparing autologous tissue adhesive using a relativelycomplex system based on ethanol precipitation has been described byWeis-Fogh⁷. Because none of these methods has yet produced a clearlysuperior autologous sealant, research in the past five years hascontinued on several approaches including plasma sealant⁸, ethanolprecipitation⁹ and rapid cryoprecipitation¹⁰. Prior patents arediscussed in later sections, Autologous Precipitate Sealants andAutologous Plasma Sealants.

None of these methods is readily adaptable for convenient use of anautologous plasma fraction as an adhesive which can be prepared quicklyduring the surgical procedure. All of the approaches suggested forpreparation of the fibrinogen containing fraction for this purpose aretoo time-consuming and complex to be finished in a short enough time tobe accomplished during the surgery. Also, in some procedures, such ascryoprecipitation, special equipment, such as refrigerated centrifuges,is required. While the prior art approach is to prepare the compositionin advance, this immediately imposes the necessity for additionalprocedures for identification and retrieval of the samples matched withthe patient, and the concomitant opportunity for error, besides theinconvenience to the patient, who must then arrange time for anadditional medical appointment. And, of course, this practice is notpossible when the surgery is conducted on an emergency basis.

Several useful reviews on surgical glues generally and on fibrin gluesparticularly have been published (Sierra, D. H. "Fibrin sealant adhesivesystems: a review of their chemistry, material properties and clinicalapplications." J Biomater Appl 7 (4 1993): 309-52; Wiseman, David M.,David T. Rovee, and Oscar M. Alverez. "Wound Dressings: Design and Use."In Wound Healing: Biochemical & Clinical Aspects, ed. I. Kelman Cohen,Robert F. Diegelmann, and William J. Lindblad. 562-580. 1st ed., Vol.Philadelphia: W. B. Saunders Company, 1992). A review of devices used bysurgeons discusses fibrin glue in context of both hemostatic agents andadhesive agents (Edlich, Richard F., George T. Rodehearer, and John G.Thacker. "Surgical Devices in Wound Healing Management." In WoundHealing: Biochemical & Clinical Aspects, ed. I. Kelman Cohen, Robert F.Diegelmann, and William J. Lindblad. 581-600. 1st ed., Vol.Philadelphia: W. B. Saunders Company, 1992). A review of the role offibrin and fibrinogen in blood coagulation has also been published(Jackson, C. M. and Y. Nemerson. "Blood coagulation." Annu Rev Biochem49 (811 1980): 765-811). Other reviews, in reverse chronological order,include:

(1) Lerner, R. and N. S. Binur. "Current status of surgical adhesives."J Surg Res 48 (2 1990): 165-81.

(2) Gibble, J. W. and P. M. Ness. "Fibrin glue: the perfect operativesealant?" Transfusion 30 (8 1990): 741-7.

(3) Thompson, D. F., N. A. Letassy, and G. D. Thompson. "Fibrin glue: areview of its preparation, efficacy, and adverse effects as a topicalhemostat." Drug Intell Clin Pharm 22 (12 1988): 946-52.

Biological Precipitate Sealants

The vast majority of prior methods for preparing blood derived glues orsealants use fibrinogen precipitated (usually cryoprecipitated) frompooled plasma, a method first published in 1972¹¹. Most biologicalsealant patents are improvements on cryoprecipitation. Most go through alyophilization step before application. A few touch on plasma, but nonementions a composition derived from concentrated plasma.

A composition from cryoprecipitation method was patented by Immuno AG¹².Since the basic method had been published previously, the compositionclaims were precisely descriptive:

Exemplary Claim: 1. A lyophilized tissue adhesive of mammalian proteinorigin which comprises fibrinogen, albumin, factor XIII, cold-insolubleglobulin and plasminogen-activator inhibitor or plasmin inhibitorwherein the fibrinogen is present in at least 33% by weight, the ratioof factor XIII to fibrinogen, expressed in units of factor XIII per gramof fibrinogen is at least 80; and fibrinogen and albumin are present ina ratio of 33 to 90:5 to 40.

Several inventions have improved on early cryoprecipitation methods. Avariation of cryoprecipitation is claimed to produce material superiorfor patients with blood coagulation disorders¹³. Stroetmann overcameproblems by mixing fibrinogen, thrombin and protease inhibitor inlyophilized form for administration as a powder¹⁴. Behring found a wayto improve solubility of (poorly soluble) cryoprecipitate¹⁵. Frenchscientists have improved the traditional cryoprecipitation by using adouble cold ethanol precipitation¹⁶. Epstein has a patent applicationfor precipitation with polyethylene glycol (only the sister (device)patent has issued)¹⁷. Cryolife has crafted a patent with a broadexemplary claim¹⁸ (quoted in note), but all the claims are limited byuse of a precipitation step.

Sierra's group has patented compositions with collagen added tofibrinogen. The notion is that collagen improves the mechanicalproperties of the sealant¹⁹. Related approaches are addition ofhyaluronic acid to make the solution more viscous before gelation(patent application)²⁰ and addition of silk fibroin for mechanicalstrength²¹.

A disclosed World patent application shows that Baxter is usingfibrinogen affinity chromatography to get around the problems withprecipitation²². A disclosed European patent application shows thatSquibb scientists have developed a method eliminating the need forthrombin²³. They use fibrin made by passing fibrinogen through a columnwith immobilized snake-venom; chaotropic agents prevent gelation.According to this work, "(f)ibrin I monomer is preferred because it can,in contrast to fibrinogen, readily be converted to fibrin polymerwithout the use of thrombin or factor XIII." From blood, the methodyields 60-90% fibrin monomer. It is activated by calcium and a pHchange.

The majority of biological patents assume a precipitation step in themanufacture of the tissue sealant.

Autologous Preoipitate Sealants

As discussed above, it is widely recognized that autologous bloodproducts are superior for safety and biocompatibility reasons alone.Columbia University has patented the use of autologous fibrin glue²⁴,but the claims are restricted to cryoprecipitate. A European patentapplication makes similar claims²⁵, but again the claims are restrictedto cryoprecipitate.

Autologous Plasma Sealants

Alterbaum has patented using small quantities of patient plasma to maketissue glue²⁶, but the plasma is not concentrated and the claims arerestricted to use of flat pack centrifugation for isolation of theplasma.

1. A method for use in the autologous preparation of fibrin glue whereina patient's blood is separated in a centrifuge having cylindrical cupspivotally mounted to a rotor to obtain plasma and wherein the plasma isseparated in the centrifuge to produce concentrated fibrinogen,comprising the steps of: (a) transferring the blood or plasma into asubstantially flat packet; (b) fixing the packet containing the blood orplasma in a recess of a substantially cylindrical insert fixtureassembly; (c) inserting said cylindrical-shaped insert fixture assemblyin a cup of the centrifuge having a complementary cylindrical shape sothat the insert fixture assembly is held snugly within said centrifugecup; (d) centrifuging the blood or plasma contained within said packetfixed in said insert fixture assembly held in said centrifuge cup toseparate the blood or plasma into components; and (e) forming fibringlue from one of said separated components.

Although no claim mentions or suggests concentrating the plasma, thepatent which appears to be most similar to the present invention (DennisGalanakis, SUNY Stony Brook) covers unconcentrated plasma glue²⁷ :

1. A method of treating with autologous mammalian plasma fibrin toaffect hemostasis, comprising the steps of: (a) obtaining a sample ofblood from said animal; (b) substantially immediately separating thewhole plasma from said blood obtained in step (a); and (c) contactingsaid whole plasma resulting from step (b) with thrombin in aphysiologically acceptable solution at a rate and in a volume at thesite of treatment to provide fibrin coagulation at said site.

Applicators

Application of fibrin glue requires mixing the two components undercontrolled conditions because gelation can be very rapid. The earliestapplicator patents are assigned to Immuno AG. The principal claimsrevolve around different syringe diameters to limit dilution of thefibrinogen component by using a smaller volume of more highlyconcentrated thrombin²⁸ and the use of medical gas to keep the syringeapplicator/mixer clear of gel²⁹. Galanakis described an application inwhich the two liquids do not mix internally but are applied side-by-sidefrom tubes that run in parallel all the way to the point ofapplication.²⁷ Very similar claims were made previously byMicromedics³⁰. Corus Medical has an aerosol device driven by pressurizedgas³¹. The Cryolife approach is to prevent gelation in asingle-component fibrin glue by pH inhibition of thrombin³². GordonEpstein has patented the use of a third suction tip to dry the surfacebefore application¹⁷.

Plasma Separators

Haemonetics' "bowl" technology allows separation of plasma continuouslyby a method which differs from the methods of the present inventiondisclosed below³³. Several patents address isolation of platelets fromblood using centrifuge sedimentation methods³⁴ or red cell barriers³⁵,but none claims an approach similar to the ones described hereinincluding sintered (porous) plastic or in which the cells pass through abarrier, leaving platelet-rich plasma behind.

Dextrans

Pharmacia has basic patents on dextranomers³⁶ and on their use in woundhealing.³⁷ Interestingly, concentration of proteins (includingfibrinogen) in the vicinity of the wound by differential absorption ofwater and electrolytes is identified as a benefit of the technology, butthe claims are limited to methods in which the dextranomer beads areapplied to the wound.

Other Patents of Interest

Fibrin sealants have been claimed as a component in a composition torepair cartilage³⁸, to help in eye surgery³⁹ and with heat-meltedcollagen for "tissue welding⁴⁰." Another approach has been to usefibrinogen/thrombin powder to coat a biodegradable fiber to stoppuncture bleeding⁴¹, or in a collagen sponge or mat with powdered fibrinsealant⁴². Addition of collagenase to fibrin sealant to improve nervehealing has been patented by Wehling⁴³. Pharmacia has patented the useof hyaluronic acid to prevent adhesions⁴⁴.

Summary of Current Use of Fibrin Sealants in the United States

Although wounds heal naturally, medicine seeks to improve and speed thewound healing process. The body's blood clotting and tissue repairmechanisms are complex cascades progressing from blood proteins (e.g.fibrinogen) to specialized repair cells (fibroblasts). Sutures, surgicalstaples or surgical adhesives hold the damaged tissues together so thathealing can progress. Surgical "fibrin sealants," sometimes called"fibrin glues," made up of human fibrinogen activated by bovine thrombinare used extensively in Europe. Such fibrin sealants have been shown tobe superior to synthetic adhesives or traditional surgery in manysituations. In addition they reduce the need for blood transfusions.Academic surgical opinion on fibrin sealants is very favorable (see thereviews cited above).

In current practice, fibrin sealant is made by isolating a concentrateof human fibrinogen, fibronectin and factor XIII, usually bycryoprecipitation, and combining it immediately before use with bovinethrombin. The thrombin converts the fibrinogen to fibrin which then gelsrapidly to form a transient hemostatic plug. The most common method ofapplication of fibrin sealant is by mixing of concentrated fibrinogenfrom pooled human blood with bovine thrombin and calcium immediatelybefore use.

Fibrin sealant is not available in the U.S. commercially, but is inEurope. The FDA is reluctant to approve these products in the UnitedStates because of the potential for virus transmission (e.g., HIV,hepatitis) from pooled human blood and the potential for immunologicalreactions. These FDA concerns are so serious that the FDA has notapproved any fibrin sealant product, despite strong interest fromsurgeons and very favorable comparative studies in the literature.

1. Hennis, H. L., W. C. Stewart, and E. K. Jeter. "Infectious diseaserisks of fibrin glue [letter]." Ophthalmic Surg 23 (9 1992): 640.

2. Berguer, R., R. L. Staerkei, E. E. Moore, F. A. Moore, W. B.Galloway, and M. B. Mockus. "Warning: fatal reaction to the use offibrin glue in deep hepatic wounds. Case reports." J Trauma 31 (3 1991):408-11.

Berruyer, M., J. Amiral, P. Ffrench, J. Belleville, O. Bastien, J.Clerc, A. Kassir, S. Estanove, and M. Dechavanne. "Immunization bybovine thrombin used with fibrin glue during cardiovascular operations.Development of thrombin and factor V inhibitors." J Thorac CardiovascSurg 105 (5 1993): 892-7.

3. Sanal, M. "Does fibrin glue cause foreign body reactions? [letter]."Eur J Pediatr Surg 3 (3 1993): 190. Sanal, M., H. Dogruyol, A. Gurpinar,and O. Yerci. "Does fibrin glue cause foreign body reactions?" Eur JPediatr Surg 2 (5 1992): 285-6.

4. Thompson, D. F., Letassy, N. A., and Thompson, G. D., Fibrin glue: areview of its preparation, efficacy, and adverse effects as a topicalhemostat. Drug Intell Clin Pharm, 1988. 22(12): p. 946-52.

5. Siedentop, K. H., D. M. Harris, and B. Sanchez. "Autologous fibrintissue adhesive." Laryngoscope 95 (9 Pt 1 1985): 1074-6.

6. Epstein, G. H., R. A. Weisman, S. Zwillenberg, and A.D. Schreiber. "Anew autologous fibrinogen-based adhesive for otologic surgery." Ann OtolRhinol Laryngol 95 (1 Pt 1 1986): 40-5.

7. Weis-Fogh, U.S. "Fibrinogen prepared from small blood samples forautologous use in a tissue adhesive system." Eur Surg Res 20 (5-6 1988):381-9.

8. Hartman, A. R., D. K. Galanakis, M. P. Honig, F. C. Seifert, and C.E. Anagnostopoulos. "Autologous whole plasma fibrin gel. Intraoperativeprocurement." Arch Surg 127 (3 1992): 357-9.

9. Kjaergard, H. K., Fogh Us Weis, and J. J. Thiis. "Preparation ofautologous fibrin glue from pericardial blood." Ann Thorac Surg 55 (21993): 543-4.

Kjaergard, H. K., U.S. Weis-Fogh, H. Sorensen, J. Thiis, and I. Rygg. "Asimple method of preparation of autologous fibrin glue by means ofethanol." Surg Gynecol Obstet 175 (1 1992): 72-3.

10. Casali, B., F. Rodeghiero, A. Tosetto, B. Palmieri, R. Immovilli, C.Ghedini, and P. Rivasi. "Fibrin glue from single-donation autologousplasmapheresis." Transfusion 32 (7 1992): 641-3.

Moretz, W, Jr., J Shea Jr., J. R. Emmett, and J Shea. "A simpleautologous fibrinogen glue for otologic surgery." Otolaryngol Head NeckSurg 95 (1 1986): 122-4.

11. Matras, Helene, H. P. Dinges, H. Lassmann, and B. Mamoli. "Zurnahtlosen interfaszikularen Nerventransplantation im Tierexperiment."Wein Med Woschtr 122 (37 1972): 517-523.

First clinical results: Kuderma, H. and Helene Matras. "Die klinischeAnwendung der Klebung yon Nervenanastomosen mit Gerinnungssubstanzen beider Rekonstruction verletzter peripherer Nerven." Wein Klin Wochenschr87 (15 1975): 495-501.

12. Schwarz, O., Linnau, Y., Loblich, F., and Seelich, T., Tissueadhesive; freeze dried wound healing agent containing Fibrinogen,albumin, factor XIII, globulin, and plasminogen blocking Agent. U.S.Pat. No. 4,414,976 (1983); Schwarz, O., Linnau, Y., Loblich, F., andSeelich, T., Tissue adhesive; factor viii, proteins, fibrinogen,globulin, albumin and plasmin inhibitor. U.S. Pat. No. 4,377,572 (1982);Schwarz, O., Linnau, Y., Loblich, F., and Seelich, T., Tissue adhesive;fibrinogen, factor XIII, albumin, plasmin inhibitor. U.S. Pat. No.4,362,567 (1982); Schwarz, O., Linnau, Y., Loblich, F., and Seelich, T.,Tissue adhesive; blood proteins; surgery. U.S. Pat. No. 4,298,598(1981); assigned to Immuno AG.

13. Martinowitz, U. and Bal, F., Improved tissue glue prepared by usingcryoprecipitate. EP (application) Patent 534,178 (1993) Assigned toOctapharma AG.

14. Stroetmann, M., Enriched plasma derivative for advancement of woundclosure and healing. U.S. Pat. No. 4,427,650 (1984); Stroetmann, M.,Fibrinogen-containing dry preparation, manufacture and use thereof. U.S.Pat. No. 4,442,655 (1984); Stroetmann, M., Enriched plasma derivativefor enhancement of wound closure and coverage. U.S. Pat. No. 4,427,651(1984); assigned to Serapharm Michael Stroetmann DE.

15. Fuhge, P., Heimberger, N., Stohr, H.-A., and Burk, W., ReadilyDissolvable Lyophilized Fibrinogen Formulation. U.S. Pat. No. 4,650,678(1987) Assigned to Behringwerke Aktiengesellschaft.

16. Burnouf-Radosevich, M. and Burnouf, T., Concentrate of thrombincoagulable proteins, the method of obtaining same and therapeutical usethereof. U.S. Pat. No. 5,260,420 (1993) Assigned to Centre Regional deTransfusion Sanguine de Lille.

17. Epstein, G. H., Method and apparatus for preparing fibrinogenadhesive from whole blood. U.S. Pat. No. 5,226,877 (1993).

18. Morse, B. S., Carpenter, J. F., Turner, A.D., and Cryolife, I.,Preparation of fibrinogen/factor XIII precipitate. U.S. Pat. No.5,030,215 (1991) Assigned to Cryolife. Claim 1: A system for collectinga blood coagulation factor comprising a first container means forreceiving whole blood, said first container means having an upper end, alower end, at least one inlet port, add at least one outlet port; firstconduit means for conveying whole blood to said first container means,said first conduit means having an end thereof coupled to a said inletport of said first container means; second container means for receivingplasma which has been separated from red blood cells in said firstcontainer means, said second container means having an upper end, thecontainer means having a first, relatively wide diameter portionadjacent to said upper end thereof, a second relatively narrow portiondefined below said first portion for receiving a blood coagulationfactor precipitate from the plasma within said second container meansand a third relatively wide portion defined below said second,relatively narrow portion so that said second, relatively narrow portiondefines a relatively narrow passage for precipitate blood coagulationfactor from said first portion to said third portion; and second conduitmeans having a first end thereof coupled to a said outlet port of saidfirst container means and a second end thereof coupled to a said inletport of said second container means for conveying plasma from said firstcontainer means to said second container means.

19. Sierra, D. H., Brown, D. M., and Luck, E. E., Surgical adhesivematerial. U.S. Pat. No. 5,290,552 (1994) Assigned to Matrix PharmInc/Project Hear.

20. Wadstroem, J., Tissue treatment composition comprising fibrin orfibrinogen and biodegradable and biocompatible polymer. WO Patent92/22,312 (1992) .

21. Iwatsuki, M. and Hayashi, T., Silk-fibroin and human-fibrinogenadhesive composition. U.S. Pat. No. 4,818,291 (1989) Assigned toAjinomoto Co Inc.

22. Tse, D. C., Alpern, M., Enomoto, S. T., Garanchon, C. M., Liu, S.L., Mankarious, S. S., and Thomas, W. R., Topical fibrinogen complex. WOApplication Patent 05067 (1993) Assigned to Baxter Int Inc.

23. Edwardson, P. A. D., Fairbrother, J. E., Gardner, R. S.,Hollingsbee, D. A., and Cederholm-Williams, S. A., Fibrin sealantcompositions and method for utilizing same. EP (Application) Patent592,242 (1993) Assigned to Squibb.

24. Rose, E. and Dresdale, A., Method of preparing a cryoprecipitatedsuspension and use thereof. U.S. Pat. No. 4,928,603 (1990) Assigned toColumbia University. Claim 1: A method of preparing a cryoprecipitatedsuspension containing fibrinogen and Factor XIII useful as a precursorin the preparation of a fibrin glue which consists essentially of: (a)freezing fresh frozen plasma from a single donor which has been screenedfor blood transmitted diseases at about -80* C. for at least 6 hours,(b) raising the temperature of the frozen plasma so as to form asupernatant and a cryoprecipitated suspension containing fibrinogen andFactor XIII, and (c) recovering the cryoprecipitated suspension.

Rose, E. and Dresdale, A., Fibrin adhesive prepared as a concentratefrom single donor fresh frozen plasma. U.S. Pat. No. 4,627,879 (1986)Assigned to Columbia University. Summary: Prodn. of a fibrin gluecomprises preparing a cryoprecipitated suspension contg. fibrinogen andFactor XIII by freezing fresh frozen plasma from a single donor whichhas been screened for blood transmitted diseases at -80 deg. C for atleast 6 hrs., raising the temp. to form a supernatant andcryoprecipitated suspension contg. fibrinogen and Factor XIII, andrecovering the suspension. A defined vol. of the suspension is appliedto the desired site and a compsn. contg. thrombin is applied to causethe fibrinogen in the suspension to be converted to the fibrinogen glue,which then solidifies as a gel.

25. Weis-Fogh, U., A Method and an Apparatus for preparing tissue repairpromoting substances. WO (Application) Patent 88/02259 (1988).

26. Alterbaum, R., Method and apparatus for use in preparation offibrinogen from a patient's blood. U.S. Pat. No. 4,714,457 (1987). Claim1: A method for use in the autologous preparation of fibrin glue whereina patient's blood is separated in a centrifuge having cylindrical cupspivotally mounted to a rotor to obtain plasma and wherein the plasma isseparated in the centrifuge to produce concentrated fibrinogen,comprising the steps of: (a) transferring the blood or plasma into asubstantially flat packet; (b) fixing the packet containing the blood orplasma in a recess of a substantially cylindrical insert fixtureassembly; (c) inserting said cylindrical-shaped insert fixture assemblyin a cup of the centrifuge having a complementary cylindrical shape sothat the insert fixture assembly is held snugly within said centrifugecup; (d) centrifuging the blood or plasma contained within said packetfixed in said insert fixture assembly held in said centrifuge cup toseparate the blood or plasma into components; and (e) forming fibringlue from one of said separated components.

27. Galanakis, D. K., Method of preparing autologous plasma fibrin andapplication apparatus therefore. U.S. Pat. No. 5,185,001 (1993) Assignedto Univ New York State Res Found.

28. Eibl, J., Habison, G., Redl, H., and Seelich, T., Arrangement forapplying a tissue adhesive. U.S. Pat. No. 4,735,616 (1988) Assigned toImmuno AG.

29. Redl, H. and Habison, G., Apparatus for Applying a tissue adhesive.U.S. Pat. No. 4,631,055 (1986) Assigned to Immuno AG.

30. Miller, C. H., Altshuler, J. H., and Arenberg, I. K., Fibrin gluedelivery system. U.S. Pat. No. 4,874,368 (1989) Assigned to Micromedics.

31. Avoy, D. R., Fibrinogen dispensing kit. U.S. Pat. No. 4,902,281(1990) Assigned to Corus Med Corp.

32. Morse, B. S., McNally, R. T., and Turner, A. D., Fibrin sealantdelivery method. U.S. Pat. No. 5,219,328 (1993) Assigned to Cryolife.

33. Headley, T. D., Plasmapheresis centrifuge bowl; disposable. U.S.Pat. No. 4,983,158 (1991) Assigned to Haemonetics Corp; Pages, E.,Disposable centrifuge bowl for blood processing. U.S. Pat. No. 4,943,273(1990); Latham, A., Jr., Apparatus for separating blood into componentsthereof. U.S. Pat. No. 4,303,193 (1981); Latham, A., Jr., Process forpheresis procedure and disposable plasma. U.S. Pat. No. 4,204,537(1980); Latham, A., Jr., Process for pheresis procedure and disposablepheresis bowl therefor. U.S. Pat. No. 4,059,108 (1977); assigned toHaemonetics.

34. Pall, D. B. and Gsell, T. C., Method for obtaining platelets. U.S.Pat. No. 5,258,126 (1993) Assigned to Pall Corp; Pall, D. B. and Gsell,T. C., Blood collection and processing system. U.S. Pat. No. 5,100,564(1992) Assigned to Pall Corp.

35. Pall, D. B., Gsell, T. C., Matkovich, V. I., and Bormann, T., Systemand method for processing biological fluid. U.S. Pat. No. 5,217,627(1993) Assigned to Pall Corp; Pall, D. B., Gsell, T. C., and Muellers,B. T., Method for processing blood for human transfusion. U.S. Pat. No.5,152,905 (1992) Assigned to Pall Corp; Eldegheidy, M. M., Automaticliquid component separator. U.S. Pat. No. 4,639,316 (1987) Assigned toBecton Dickinson & Co.

36. Process for the Manufacture of Hydrophilic High Molecular WeightSubstances from Dextran Substances. GB Patent 854,715 (1965); Gelotte,E. B. and Soderquist, B. G. F., [Hydroxy compound copolymersdextran-epichlorhydrin] Verfahren zur Herstellung von Substitutionsprodukten von Mischpolymerisaten. DD Patent 56,103 (1965); Beil,W., Hoeppner, A., Wolff, H. J., and Beil, H., [Verfahren zur Herstellungyon hochmolecularen hydrophilen Vernetzungsproducten von Polysaccharidenoder deren Derivaten oder yon Polyvinylalkohol in Form yon Gelkoernern]High mol. wt. hydrophilic copolymer of a hydroxy group-contng.,non-ionic polymer is obtd. in the form of gel grains by reacting thepolymer, in the presence. DE Patent 1,443,359 (1962); assigned toPharmacia.

37. Rothman, U. S. and Jacobsson, S. A., Method for cleansing fluiddischarging skin surfaces, wounds and mucous membranes and means forcarrying out the method; dry particles of water-insoluble swellablepolymer. U.S. Pat. No. 4,537,767 (1985) Assigned to Pharmacia AB;Rothman, U. S. and Jacobsson, S. A., Method for cleansing fluiddischarging skin surfaces, wounds and mucous membranes and means forcarrying out the method. U.S. Pat. No. 4,225,580 (1980) Assigned toPharmacia AB.

38. Hunziker, E. B., Method and compositions for the treatment andrepair of defects or lesions in cartilage. U.S. Pat. No. 5,206,023(1993) .

39. Sarfarazi, F., Sarfarazi method of closing a corneal incision. U.S.Pat. No. 5,190,057 (1993); O'Donnell, F., E., Jr., Maremen, E., andNalbandian, R. M., Intraocular lens implant and method of locating andadhering within the posterior chamber. U.S. Pat. No. 5,002,571 (1991).

40. Sawyer, P. N., Collagen welding rod material for use in tissuewelding. U.S. Pat. No. 5,156,613 (1992) Assigned to Interface BiomedicalLaboratories.

41. Sakamoto, I., Unigame, T., and Takagi, K., Hemostatic agent. U.S.Pat. No. 4,655,211 (1987) Assigned to Unitika KK.

42. Zimmermann, E. and Schiele, U., Agent for sealing and healingwounds. U.S. Pat. No. 4,453,939 (1984) Assigned to Hormon-Chemie Munch;Stemberger, A., Gewebeverklebarre kollagene Wundauflage. EP Patent102,773 (1983) Assigned to Dr. Ruhland Nachf. GmbH.

43. Wehling, P., Method of enhancing the regeneration of injured nervesand adhesive pharmaceutical formulation therefor. U.S. Pat. No.5,279,825 (1994) Assigned to Advanced Biofactures; Wehling, P., Methodof enhancing the regeneration of injured nerves and adhesivepharmaceutical formulation therefor; supplying collagenase to the zoneof nerve injury during regeneration. U.S. Pat. No. 5,173,295 (1992)Assigned to Advanced Biofactures.

44. Lindblad, G. and Buckley, P., Composition and method for preventionof adhesions between body tissues. U.S. Pat. No. 5,190,759 (1993)Assigned to Kabi Pharmacia AB.

SUMMARY OF THE INVENTION

Accordingly, one object of the present invention is to provide a novelmethod and apparatus for rapidly preparing a plasma concentrate fromblood containing fibrinogen, prothrombin (the inactive precursor ofthrombin) and other blood proteins, and platelets (thrombocytes)(citrate, heparin or other anticoagulant may be present in an amountsufficient to prevent initiation of clotting).

A further object of the present invention is to provide a novel methodand apparatus for rapidly preparing a plasma concentrate from apatient's own blood (autologous tissue sealant).

A further object of the present invention is to provide a novel methodand apparatus for rapidly preparing a concentrate from a patient's ownblood while in the operating room (star autologous tissue sealant).

A further object of the present invention is to provide a compositionwith physical, chemical and biological properties superior to plasmaprotein precipitate derived tissue sealants.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the invention and many of the attendantadvantages thereof will be readily obtained as the same becomes betterunderstood by reference to the following detailed description whenconsidered in connection with the accompanying drawings, wherein:

FIG. 1 shows an embodiment of a disposable cartridge in accordance withthe present invention, employing plasma separation by open cellhydrophobic foam and plasma concentration by open cell foam with beadsheld thereto by electrostatic force;

FIG. 2 shows an embodiment of a disposable cartridge in accordance withthe present invention, employing plasma separation by sintered plasticbarrier and plasma concentration by beads distributed in a trough;

FIG. 3 shows an embodiment of a disposable cartridge in accordance withthe present invention, employing plasma separation by annular chamberand plasma concentration by open cell foam with beads held thereto byelectrostatic force;

FIG. 4 shows an embodiment of a disposable cartridge in accordance withthe present invention, employing plasma separation by felt mat andplasma concentration by beads impregnated into plastic discs;

FIG. 5 depicts a single enlarged concentrator bead in a solution of lowmolecular weight and macromolecule components (e.g., plasma) inaccordance with the present invention; and

FIG. 6 shows an embodiment of a disposable cartridge in accordance withthe present invention after centrifugation and during syringe withdrawalof the plasma concentrate;

FIG. 7 shows an embodiment of the applicator in accordance with thepresent invention employing coaxial needles to allow mixing of the twocomponents;

FIG. 8 shows an embodiment of the applicator in accordance with thepresent invention employing an activator cartridge to allow applicationof single component fibrin sealant; and

FIG. 9 graphically shows the relationship between the concentration ofproteins and their molecular weight, for a protein solution orsuspension concentrated 3× with beads having a molecular weight cutoffof 10 kDa in accordance with the present invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

An inexpensive device with a disposable cartridge for preparation oftissue sealant is disclosed. The device is particularly applicable tostat preparation of autologous tissue sealant. The disposable cartridgemay fit in the palm of the hand and is hermetically sealed to eliminatepossible exposure to patient blood and ensure sterility. Methods ofsealing tissue in which the tissue sealant is applied immediately aftermixing platelet-rich plasma concentrate (from the device) with asolution of calcium and thrombin or in which the tissue sealant isapplied immediately after physical or immobilized enzyme activation aredisclosed.

Preparation in the operating room of 5 cc sealant from 50 cc patientblood requires less than 15 minutes and only one simple operator step.There is no risk of tracking error because processing can be done in theoperating room. Additional chemicals used in the plasma sealant andpreparation thereof may be limited to anticoagulant (for example,citrate) and calcium salts (e.g., chloride).

Adhesive and tensile strengths are comparable to pooled blood fibrinsealants that are available in Europe commercially. Antifibrinolyticagents (such as aprotinin) are not necessary because the tissue sealantcontains high concentrations of natural inhibitors of fibrinolysis fromthe patient's blood. The tissue sealant also contains patient plateletsand additional factors not present in available fibrin sealants thatpromote wound healing.

The present (autologous) tissue sealant gels rapidly as it is mixed withcalcium (and optionally bovine or human thrombin) while being applied tothe wound. This sealant contains only FDA-approved or approvablecomponents. In the present invention, a "tissue sealant" contains thecomponents found in blood that contribute to clot formation, tissueadhesion and wound healing (preferably all such components in blood),and thus, is distinguished from prior "fibrin sealants" which typicallycontain only cryoprecipitated proteins and no platelets.

The present invention concerns a method of making platelet-rich plasma(PRP) concentrate, which can then be combined with calcium/thrombin tomake tissue sealant. The resulting gel is a composition that has neverbefore been described in the scientific or patent literature.

Referring now to the drawings, wherein like reference numerals designateidentical or corresponding parts throughout the several views, and moreparticularly to FIG. 1 thereof, in a preferred embodiment of the presentinvention, citrated plasma is processed in two stages in a disposablecentrifuge cartridge. In a first stage, platelet-rich plasma isseparated from cells when centrifugal force causes red and white cellsto lodge irreversibly in a first separator (e.g., a hydrophobic opencell foam). In a second stage, the platelet-rich plasma is concentratedby contact with a concentrator (e.g., dextranomer beads) that absorbswater, electrolytes and small proteins, leaving a platelet-rich plasmaconcentrate. Thus, the present method of making platelet-rich plasmacomprises the steps of:

(a) separating plasma and platelets from whole blood;

(b) contacting the plasma and platelets with a concentrator to providethe concentrated platelet-rich plasma; and

(c) separating the concentrated platelet-rich plasma from theconcentrator.

Preferably, the cartridge is disposable and sealed (e.g., see housing 1in each of FIGS. 1 through 4), making contamination impossible. Wherethe method is performed in a sealed cartridge, the separating step (a)above may be conducted, for example, in a first chamber of the cartridgewhich is in fluid communication with the blood fill port (inlet). Theplasma and platelets may then be contacted with a concentrator (e.g., ina second chamber of the cartridge in fluid communication with the firstchamber) sufficiently to concentrate the plasma. An advantage of thesealed, disposable cartridge is that it can be used in the operatingroom, thus eliminating the possibility of tracking errors andcontamination.

In a further embodiment, the method is one for making autologousconcentrated platelet-rich plasma, in which the method above for makingconcentrated platelet-rich plasma further comprises, prior to theseparating step (a), collecting blood from a patient and adding aneffective mount of citrate to inhibit coagulation (in accordance withstandard methods), and injecting a standard quantity of blood (e.g., 50cc) into the blood fill port (inlet) 5 of a cartridge 1.

Plasma is separated from blood by a combination of conventionalcentrifugation technology and inventions for ease of separation of thepacked cells from the plasma. Centrifuges which are capable of spinningat variable speeds up to 3,000-10,000 rpm in either direction and whichare capable of being controlled by a microprocessor (i.e., for spinningat predetermined rates of rotation for predetermined lengths of time)are known and are easily adapted, if necessary, for use with the presentapparatus for separating and concentrating plasma. Following injectionof the whole blood, the operator starts the microprocessor-controlledcentrifuge by push-button. All remaining steps until removal of theconcentrate may be automatic.

Although cells are conventionally separated from plasma by theirproperty of packing into a cell mass during centrifugation (thentypically aspirated), the cartridges are designed to effect rapidseparation of the plasma into the next, concentrator chamber. In oneembodiment (FIG. 1), red and white cells remain trapped in a hydrophobicopen cell foam 14. In another embodiment (FIG. 4) red and white cellsremain trapped in a felt annulus 7. In a third embodiment (FIG. 2) thecells pass through sinuous pathways in a sintered annulus 8 and remaintrapped outside. In a fourth embodiment (FIG. 3) red and white cellsmigrate through a small gap 16 and remain trapped in an outer annulus bythe force of gravity when the centrifuge stops.

Optionally, the plasma may be contacted with dextranomer beads that havebeen bonded by electrostatic force with an open, hydrophobic cellularfoam 15 (in accordance with the apparatus of FIGS. 1 and 3). When wettedthe beads lose their electrostatic attraction and begin to swell.Alternatively, the plasma may be contacted with free dextranomer beads2, e.g., in accordance with the apparatus of FIG. 2. The centrifuge mayrotate briefly clockwise then counterclockwise a few times at a speedsufficient to evenly distribute the free dextranomer beads 2 outside theconcentrator cup lip 3.

The centrifuge may then spin at from 1,000 to 10,000 rpm, preferably3,000 rpm, for one to ten minutes to separate platelet-rich plasma fromcells. First, the whole blood passes through a pore 4 in the blood inlet6 into the separation chamber. Cells may become trapped in a matrix 14(as shown in FIG. 1), enmeshed in a matrix 7 (as shown in FIG. 4), orpass through sintered hydrophobic plastic or other porous material 8 (asshown in FIG. 2), leaving platelet-rich plasma in the chamber.

The centrifuge may then be stopped for 15-90 seconds. Under the force ofgravity, the platelet-rich plasma passes through an annulus (or througha hydrophilic filter 9) into the second chamber 10 (the lower chamber inFIGS. 1 through 4). This chamber may also be identified as a firstconcentrator chamber.

In the first concentrator chamber, platelet-rich plasma comes intocontact with the concentrator (e.g., dextranomer or polyacrylamide),either bonded by electrostatic force with an open, hydrophobic cellularfoam 15 (FIGS. 1 and 3) or free beads 2 (FIG. 2) or beads embossed ontodisc(s) 11 (FIG. 4). Dextranomer and polyacrylamide concentrators arecommercially available (as SEPHADEX from Pharmacia and as BIO-GEL P fromBio-Rad Laboratories, respectively). Alternatively, instead ofdextranomer or polyacrylamide absorbent beads, other concentrators, suchas SEPHADEX moisture or water absorbants (available from Pharmacia),other polyacrylamide absorbants, silica gel, zeolites, dextramines,alginate gel, starch, cross-linked agarose, etc., in the form of beadsor discs are also acceptable concentrators.

In essence, any material that absorbs water (i.e., which is sufficientlydry to permit water absorption) and which does not significantlydenature the proteins essential to blood clotting (e.g., fibrinogen andfactor XIII) can be used as a concentrator, as canappropriately-selected membranes, which may also employ an osmoticgradient. Further, where the centrifuge has autoclave-like capabilities(i.e., the ability to rotate its contents at elevated temperaturesand/or atmospheric pressures other than 1 atm), mechanicalconcentrators, such as an elevated temperature (e.g., a temperature of30°-80° C., preferably 35°-60° C.) or a reduced pressure/vacuum (e.g.,0.1-500 Torr, preferably 1-200 Torr) may serve as a concentrator.However, beads employed as described both above and hereunder are thepreferred concentrator.

To maximize results, initial slow rotation of the centrifuge aids mixing(and thus contact) of the concentrator and the plasma. "Slow rotation"refers to a centrifuge speed of from 20 rpm to 500 rpm, preferably from100 rpm to 300 rpm.

The porosity and hydrophobicity of the porous wall 7, 8 or 14 are suchthat the low centrifugal force generated is sufficient to drive plasmaup the wall but is not sufficient to overcome surface tension, and theplasma remains with the compartment at the inner surface.

The beads 2 swell with water, ions, and low molecular weight proteins 51(see FIG. 5). The size of the pores 52 in the concentrator bead 2 ischosen so that the higher molecular weight proteins 53 becomeconcentrated in the space around the beads. Any commercially availablebead concentrators for concentrating proteins having a molecular weightabove a certain preselected molecular weight cutoff value may be used.

As those of ordinary skill in protein chromatography understand, a"molecular weight cutoff" refers to pores in a matrix (e.g., SEPHADEX)which are distributed about a mean size. Moieties (e.g., proteins) morethan, for example, 25-50% smaller than mean size diffuse into the beadrapidly, and moieties more than, for example, 25-50% larger than meansize do not diffuse into the bead. Proteins having a size within, forexample, 25-50% of the mean size diffuse into the bead slowly (dependingon the particular bead selected). Thus, a bead having a molecular weightcutoff of 10 kDa concentrates proteins according to molecular weight asshown graphically in FIG. 9.

Consequently, in the present application, "high molecular weightcompounds" refer to those compounds having a molecular weight greaterthan the preselected molecular weight cutoff value of the concentrator.Examples of high molecular weight proteins in plasma may includefibrinogen and factor XIII, depending on the selected molecular weightcutoff. Preferred concentrator materials include those beads ormaterials having a molecular weight cutoff value of 100 kDa, preferably30 kDa and more preferably 5 kDa.

It is important to note that the platelet-rich plasma concentratecontains all components of the original plasma, in either the originalconcentration (ions and compounds with molecular weight less than theconcentrator cutoff) or at a greater concentration thannaturally-occurring plasma (e.g., platelets and compounds with molecularweight more than the concentrator cutoff). The protein concentratecontains the same ion concentrations as the unconcentrated plasma.

The quantity of beads is chosen so that the platelet rich plasma isconcentrated by a desired amount, e.g., 3× (the approximate level ofconcentration achieved by a predetermined volume of fluid surroundingclose-packed spheres in the same volume). When using DEBRISAN(trademark, Johnson and Johnson), approximately 2.6 gm is required per10 cc plasma to achieve 3-fold concentration.

After absorption of water and low molecular weight solutes into theconcentrator, the rate of rotation is increased (e.g., to 1,000-10,000rpm, preferably to about 3,000 rpm), forcing the concentratedplatelet-rich plasma out through a filter 12 into an outer compartment.The centrifuge may then be stopped, and under the force of gravity,concentrated platelet-rich plasma may be collected in a bottom well 13.The concentrated platelet-rich plasma may be left for extended periods(e.g., hours) without spontaneous gelation or other deleterious effect.

Concentrated platelet-rich plasma, ready for use, may then be removedinto a syringe 16 as in FIG. 6.

Thus, the present invention also concerns a method of sealing tissue,comprising the steps of contacting the present concentrated plasma withan activator (either a thrombin/calcium solution or disrupted plateletsas described below) to provide an activated plasma concentrate, andapplying the activated plasma concentrate to tissue in need of sealing.

In one embodiment of the method of sealing tissue, a separately-preparedsolution containing amounts of thrombin and calcium effective tocoagulate the concentrated plasma may be combined with the concentratedplasma (in accordance with methods described in the literature forcryoprecipitated fibrinogen). Once combined, the solution andconcentrated plasma rapidly gel to form a tissue sealant. An embodimentwith features superior to any previously described is shown in FIG. 7.In the barrel of the syringe a cylindrical chamber 31 containingthrombin and calcium solution is surrounded by an annular chamber 32containing concentrated platelet-rich plasma. Plunger 30 causes thesolutions to exit the apparatus through a coaxial needle, 33 (thrombin)and 35 (concentrated plasma), and mix at the tip 36. An optional sleeve34 ensures proper mixing, and may be retracted to expose premature gelfor easy removal.

In a further optional embodiment, a needle tip designed to activatefibrinogen may be used to apply a single-component concentrate (i.e.,the concentrated plasma) without the use of added thrombin. In thisembodiment, calcium is added to the concentrate immediately prior to orduring its application to tissue in need of sealing. FIG. 8 shows asyringe 21 and applicator needle tip 23 separated by activator cartridge22 through which platelet-rich plasma passes for activation byimmobilized proteolytic enzyme, e.g., bovine thrombin.

In a further optional embodiment, a needle tip designed to disruptplatelets may be used to apply a single-component concentrate (i.e., theconcentrated plasma) without the use of thrombin. In this embodiment,calcium is added to the concentrate immediately prior to or during itsapplication to tissue in need of sealing. FIG. 8 shows a syringe 21 andapplicator needle tip 23 separated by activator cartridge 22 throughwhich platelet-rich plasma passes for activation by, e.g., nylon wool.

The present invention also concerns an apparatus for concentratingplatelet-rich plasma, comprising:

an inlet,

a first chamber in fluid communication with the inlet, containing afirst separator for separating plasma and platelets from whole blood,thus forming platelet-rich plasma,

a second chamber in fluid communication with the first chamber,containing a concentrator for concentrating the platelet-rich plasma anda second separator for separating the concentrated platelet-rich plasmafrom the concentrator, and

an outlet for withdrawing the concentrated platelet-rich plasma.

In a further embodiment, the first separator comprises an open cellhydrophobic foam which traps red blood cells and white blood cellstherein under surface tension force.

In a further embodiment, the first separator comprises a porous plasticwall which allows passage of red blood cells and white blood cellstherethrough under a centrifugal force, but does not permit passage ofred blood cells and white blood cells in the absence of the centrifugalforce.

In a further embodiment, the first separator comprises an annulus at thetop of the first chamber that allows red blood cells and white bloodcells to pass into an outer annulus and be separated from plasma,maintained by gravity when the centrifuge stops.

In a further embodiment of the apparatus, the concentrator may beembedded onto a paper disc, or onto a plurality of discs (of anycommercially available material), or may stick electrostatically or byother forces to an open cell matrix.

Many designs of a preferred embodiment of the present cartridge areenvisioned (for example, FIGS. 1 through 4). The designs of the firstand second chambers are largely independent, and may be combined into asingle cartridge at will. All four designs shown have at least oneseparation chamber in fluid connection with at least one concentrationchamber.

The most preferred embodiment, FIG. 1, uses open cell hydrophobic foam14 for separation of plasma from cells and effects plasma concentrationby beads held by electrostatic force in an open cell foam 15. Anotherpreferred embodiment, shown in FIG. 2, uses plasma separation bysintered plastic barrier 8 and plasma concentration by beads distributedin a trough 2. Another preferred embodiment, shown in FIG. 3, usesplasma separation by annular chamber 16 and plasma concentration bybeads held by electrostatic force within open cell foam 15. Anotherpreferred embodiment, shown in FIG. 4, uses plasma separation by feltmat 7 and plasma concentration by beads impregnated into plastic discs12.

Each separator can be used with each concentrator. The four separatorsmay be combined with the three concentrators to give twelve possiblearrangements for the disposable cartridge.

A. Open Cell Hydrophobic Foam Plasma Separator and Dextranomer Bead inOpen Cell Hydrophobic Foam Concentrator

In FIG. 1, the ready cartridge is shown in the right half and the pathof the liquid is shown by the dashed arrows on the left half. The entirechamber is radially symmetric except the septum 5 for the whole bloodinlet. The receiving cup 6 holds blood until centrifugal force causesblood to pass through the filter 4. In the separation chamber,centrifugal force causes cells to pass into the open cell hydrophobicfoam 14 and accumulate therein; continuing centrifugal force causescells to migrate into the annulus of open cell foam and platelet richplasma to remain within the separation chamber. The open cellhydrophobic foam 14 is a honeycomb-like hydrophobic material whichallows fluids and small particles to flow freely (e.g., open cellpolyurethane foam). Like a wetted sponge, the foam holds liquid againsta certain head of pressure due to surface tension forces. Thus, bloodcells or other suspended particulates remain entrapped within the foamwhen the centrifuge stops and separated platelet-rich plasma drains fromthe surface under the force of gravity. Foam can be either rigid orflexible and can be formed into the appropriate annular shape for thedevice by molding or die-cutting. The parts are sized so that the packedcell (e.g., erythrocyte and leukocyte) layer is fully contained withinthe outer open cell foam chamber, which retains the cells when thecentrifuge stops.

Concentration comes about when platelet-rich plasma contactsconcentrator (e.g., dextranomer) beads 2 in the concentration chamber10. These beads may be a molecular sieve used in chromatography(SEPHADEX, available from Pharmacia; BIO-GEL P, available from Bio-RadLaboratories) and debriding (DEBRISAN, available from J&J), or maycomprise silica gel, zeolites, dextramines, alginate gel, starch,cross-linked agarose, etc. DEBRISAN appears similar to SEPHADEX G-25,and both are available commercially.

The beads are held by static electric forces onto the surface of opencell hydrophobic foam 15. The open cell hydrophobic foam 15 is ahoneycomb-like hydrophobic material. After the platelet-rich plasma hasentered the concentration chamber 10, the cartridge 1 is gently rotatedto mix platelet-rich plasma with concentrator beads 2. As the fluidpenetrates the open foam 15, the beads lose their affinity for the foamand begin to swell. Foam 15 may be made from the same material as foam14 (e.g., open cell polyurethane), but may have a different porosity.The porosity of foams 14 and 15 is selected empirically on the basis ofthe sizes and/or types of particles to be retained (e.g., cells by foam14 and concentrator beads by foam 15). In commercially available opencell, three-dimensional polyurethane foams, porosity is known to becorrelated to density (which is measured in, e.g., weight per cubicfoot).

After a few minutes (e.g., 1-10 min., preferably 3-5 min.), theconcentration is complete. Note that concentration by approximatelythree times corresponds to fluid surrounding close-packed spheres; thus,three times concentration is optimal. Concentration of more than threetimes is possible, but to achieve best results, a second concentrationchamber should be used for further concentration of plasma concentratedin a first chamber. Thus, concentration of nine times is possible for athree-chamber cartridge containing two concentration chambers.

When concentration is complete, the centrifuge increases its rate ofrotation and the increased centrifugal forces cause concentratedplatelet-rich plasma to pass through the hydrophobic annulus 12.Sintered plastic 12 (e.g., POREX) is comprised of small particles ofplastic fused to create a porous structure. Fluids and smallparticulates can flow freely through the tortuous pathways defined bythe spaces between the fused plastic particles when the material is wet.If the plastic is not specially formulated or treated, the sinteredplastic will be hydrophobic and when in the dry state will affordresistance to influx of water, more greatly as the size of the pathwaysis reduced. This property of "entry pressure" allows the wetting of thesurface of a hydrophobic porous wall without substantial entry of fluidinto the material. At greater pressure, i.e., when centrifugal force isincreased, this resistive force is overcome and fluid breaks through thebarrier to enter and pass through the porous wall.

The centrifuge stops, and concentrated platelet-rich plasma runs to thebottom under the force of gravity and is ready to be removed throughfilter 13 as in FIG. 6.

Finally, note that the concentrator cup 10 is shaped like an invertedfunnel to vent the lower chamber to the upper chamber allowingdisplacement of air from the lower chamber by plasma.

B. Hydrophobic Porous Wall Plasma Separator and Free Dextranomer BeadConcentrator

In FIG. 2, the ready cartridge is shown in the right half and the pathof the liquid is shown by the dashed arrows on the left half. The entirechamber is radially symmetric except the septum 5 for the whole bloodinlet. The receiving cup 6 holds blood until centrifugal force causesblood to pass through the filter 4. In the separation chamber,centrifugal force causes cells to pass through the sinuous channels offilter 8 and accumulate in the outer annulus. The filter 8 (e.g., 80 μPOREX) is comprised of small particles of plastic fused to create aporous structure. Fluids and small particulates can flow freely throughthe tortuous pathways defined by the spaces between the fused plasticparticles when the material is wet. If the plastic is not speciallyformulated or treated, the sintered plastic will be hydrophobic and whenin the dry state will afford resistance to influx of water, more greatlyas the size of the pathways is reduced. This property of "entrypressure" allows the wetting of the surface of a hydrophobic porous wallwithout substantial entry of fluid into the material. At greaterpressure, i.e., when centrifugal force is increased (e.g., at1000-10,000 rpm or higher), this resistive force is overcome and fluidbreaks through the barrier to enter and pass through the porous wall.This leaves plasma and platelets in the chamber. The parts are sized sothat the packed cell (e.g., erythrocyte and leukocyte) layer is fullycontained within the filter and outer annulus. When the centrifuge stopsand the platelet-rich plasma passes through the hydrophilic funnelfilter 9, the force of gravity is insufficient to cause cells to passthrough the filter 8 and back into the separation chamber.

Hydrophilic funnel filter 9 separates the upper and lower compartmentsso that the loose beads cannot tumble into the upper compartment duringshipping and handling. This material might, for example, be similar tothat used for filter 8. In order that entry pressure not be so high asto preclude free drainage of plasma (by gravity) into the lowercompartment after separation from blood cells, however, this materialwould preferably be of effective porosity minimally sufficient to ensureentrapment of beads in the concentrator chamber 10. The material wouldalso most desirably be treated in a manner such as to render it morehydrophilic, (e.g., plasma or corona treated or blended or coated withsurfactant) in order to further reduce entry pressure.

Concentration comes about when platelet-rich plasma contactsconcentrator beads 2 in the concentration chamber 10. These beads may bea molecular sieve used in chromatography (SEPHADEX, available fromPharmacia; BIO-GEL P, available from Bio-Rad Laboratories) and debriding(DEBRISAN, available from J&J), or may comprise silica gel, zeolites,dextramines, alginate gel, starch, cross-linked agarose, etc. DEBRISANappears similar to SEPHADEX G-25, and both are available commercially.

The beads are sealed into the chamber, but may become unevenlydistributed during shipment. A hydrophilic filter 9 keeps the beads inthe concentrator chamber 10. Immediately after the addition of wholeblood, the cartridge may be gently rotated and counter-rotated a fewtimes to evenly distribute the beads behind the lip 3.

After the platelet-rich plasma has entered the concentration chamber 10,the cartridge 1 is gently rotated to mix platelet-rich plasma withconcentrator beads 2. For best results, it is important to keep theslurry in motion because of the tendency for protein to locallyconcentrate around each bead, retarding absorption. After a few minutes(e.g., 1-10 min., preferably 3-5 min.), the concentration is complete.Note that concentration by approximately three times corresponds tofluid surrounding close-packed spheres; thus, three times concentrationis optimal. Concentration of more than three times is possible, but toachieve best results, a second concentration chamber should be used.Thus, concentration of nine times is possible for a three-chambercartridge containing two concentration chambers.

When concentration is complete, the centrifuge increases its rate ofrotation and the increased centrifugal forces cause concentratedplatelet-rich plasma to pass through the hydrophobic annulus. Finally,the centrifuge stops, and concentrated platelet-rich plasma runs to thebottom under the force of gravity and is ready to be removed.

Finally, note that the concentrator cup 10 is shaped like an invertedfunnel to vent the lower chamber to the upper chamber allowingdisplacement of air from the lower chamber by plasma.

C. Annular Chamber Plasma Separator and Dextranomer Bead in Open CellHydrophobic Foam Concentrator

In FIG. 3, similar to FIG. 1 in many respects, the ready cartridge isshown in the right half and the path of the liquid is shown by thedashed arrows on the left half. The entire chamber is radially symmetricexcept the septum 5 for the whole blood inlet. The receiving cup 6 holdsblood until centrifugal force causes blood to pass through the filter 4.In the separation chamber, centrifugal force causes cells to pass abovethe lip 16 into an outer annular chamber and accumulate therein;continuing centrifugal force causes cells to migrate into the outerannulus and platelet rich plasma to remain within the separationchamber. The parts are sized so that the packed cell (e.g., erythrocyteand leukocyte) layer is fully contained within the outer annularchamber, which retains the cells when the centrifuge stops. When thecentrifuge stops, the platelet-rich plasma flows into the concentratorchamber 10.

Concentration comes about when platelet-rich plasma contactsconcentrator beads 2 in the concentration chamber 10. These beads are amolecular sieve used in chromatography (SEPHADEX, available fromPharmacia; BIO-GEL P, available from Bio-Rad Laboratories) and debriding(DEBRISAN, available from J&J), or may comprise silica gel, zeolites,dextramines, alginate gel, starch, cross-linked agarose, etc. DEBRISANappears similar to SEPHADEX G-25, and both are available commercially.

As in FIG. 1, the beads are held by static electric forces onto thesurface of open cell hydrophobic foam 15. The open cell hydrophobic foam15 is a honeycomb-like hydrophobic material. After the platelet-richplasma has entered the concentration chamber 10, the cartridge 1 isgently rotated to mix platelet-rich plasma with concentrator(dextranomer) beads 2. As the fluid penetrates the open foam 15, thebeads lose their affinity for the foam and begin to swell. For bestresults, it is important to keep the slurry in motion because of thetendency for protein to locally concentrate around each bead, retardingabsorption. After several minutes, the concentration is complete. Notethat concentration by approximately three times corresponds to fluidsurrounding close-packed spheres; thus, three times concentration isoptimal. Concentration of more than three times is possible, but toachieve best results, a second concentration chamber should be used.Thus, concentration of nine times is possible for a three-chambercartridge containing two concentration chambers.

When concentration is complete, the centrifuge increases its rate ofrotation and the increased centrifugal forces cause concentratedplatelet-rich plasma to pass through the hydrophobic annulus 12.Sintered plastic 12 is made of POREX, comprised of small particles ofplastic fused to create a porous structure. Fluids and smallparticulates can flow freely through the tortuous pathways defined bythe spaces between the fused plastic particles when the material is wet.If the plastic is not specially formulated or treated, the sinteredplastic will be hydrophobic and when in the dry state will affordresistance to influx of water, more greatly as the size of the pathwaysis reduced. This property of "entry pressure" allows the wetting of thesurface of a hydrophobic porous wall without substantial entry of fluidinto the material. At greater pressure, i.e., when centrifugal force isincreased (e.g., at 1000-10,000 rpm or higher), this resistive force isovercome and fluid breaks through the barrier to enter and pass throughthe porous wall.

The centrifuge stops, and concentrated platelet-rich plasma runs to thebottom and is ready to be removed through filter 13 as in FIG. 6.

Finally, note that the concentrator cup 10 is shaped like an invertedfunnel to vent the lower chamber to the upper chamber allowingdisplacement of air from the lower chamber by plasma.

D. Fibrous Matrix Plasma Separator and Dextranomer-Impregnated DiscConcentrator

FIG. 4 is similar to the previous figures in many respects. Like FIG. 1,the ready cartridge is shown in the right half and the path of theliquid by the dashed arrows on the left half. The entire chamber isradially symmetric except the septum 5 (the whole blood inlet).

Separation is done by driving cells into a felt or foam mat 7. The partsare sized so that the packed cell (e.g., erythrocyte and leukocyte)layer is fully contained within the felt or foam filter, which retainsthe cells when the centrifuge stops.

In this design, even distribution of the dextranomer beads is assured byusing plastic discs embossed with dextranomer beads. This can be doneeither with heat or solvent softening of the plastic followed by dippingin dextranomer beads or affixing beads to the plastic with adhesive. Asthe beads swell they detach from the discs. Separation of theconcentrated platelet-rich plasma is done as in the above description ofthe present method of making concentrated platelet-rich plasma.

E. Method of Manufacture of the Disposable Cartridge

The various rigid plastic components comprising the cartridge aremanufactured by injection molding or by pressure- or vacuum-forming,assembled and sealed by conventional methods (e.g., either by adhesivesor welding). From FIGS. 1 through 4 it will be apparent to one skilledin the art of plastics fabrication that manufacture is relatively simpleand inexpensive. The final assembled product is packaged, radiationsterilized and ready for distribution.

F. Plasma Concentrate, Platelet-rich plasma Concentrate and TissueSealant

The present invention also concerns a plasma concentrate, prepared bythe present process. The present plasma concentrate may beplatelet-rich, platelet-poor or platelet-free (i.e., the plateletconcentration is not limited), but is preferably platelet-rich. Theconcentration of platelets can be controlled by the sedimentation rate,in accordance with known procedures and/or known platelet sedimentationbehavior. Preferably, the plasma concentrate is autologous (administeredto the patient from whom the whole blood was taken).

In one embodiment, the present plasma concentrate comprises platelets,from 5 to 400 mg/ml of fibrinogen, from 0.5 to 35 mg/ml of fibronectin,and a physiologically acceptable carrier comprising water andphysiologically acceptable inorganic and organic ions in aphysiologically acceptable concentration. In a preferred embodiment, theplasma concentrate is prepared from whole blood, and platelets areconcentrated at least one-and-one-half times relative to theconcentration of platelets in unconcentrated plasma from the same wholeblood.

The present plasma concentrate may further contain a compound selectedfrom the group consisting of an antibiotic, a collagen fleece,collagenase, hyaluronic acid, a wound-healing factor, zinc ions in anamount effective to decrease coagulation time of said plasma, and abiologically acceptable dye. A preferred biologically acceptable dye isdisulphine blue, contained in an amount sufficient to visibly detect theplasma concentrate during its application to tissue. In a preferredembodiment, the plasma concentrate further comprises a pharmaceuticallyactive compound, particularly an antibiotic to reduce the probability ofinfection by extraneous microorganisms.

The present invention also concerns a tissue sealant, comprising thepresent plasma concentrate and an activator. Preferably, the plasmaconcentrate in the present tissue sealant is platelet-rich plasmaconcentrate.

In one embodiment of the present tissue sealant, the activator is amixture of thrombin and calcium in amounts sufficient to permit gelationof the plasma concentrate. In a further embodiment, the activator isautologous thrombin and platelets.

G. Apparatus for and Method of Mixing Two-Component Fibrin Sealant

Parallel syringes with the two components (e.g., concentratedplatelet-rich plasma and calcium/thrombin solution) converge on acoaxial needle with mixing of the components at the application site,thus avoiding premature gelation; see FIG. 7. In the barrel of thesyringe a cylindrical chamber 31 containing thrombin and calciumsolution is surrounded by an annular chamber 32 containing concentratedplatelet-rich plasma. Plunger 30 causes the solutions to exit theapparatus through coaxial needles and mix at the tip 36. An optionalsleeve 34 ensures proper mixing, and may be retracted to exposepremature gel for easy removal.

H. Dispenser/Activator for Platelet-Rich Plasma Concentrate

Most users of fibrin sealants have resorted to the use of bovinethrombin to induce gelation. In a preferred embodiment of the presentinvention, it is desirable to avoid excessive use of bovine proteins inthe final preparation. Thus, a method and apparatus employing a smallamount of bovine or other thrombogenic enzyme to trigger theautocatalytic activation of the coagulation process is envisioned. FIG.8 shows a syringe 21 and applicator needle tip 23 separated by activatorcartridge 22 through which platelet-rich plasma passes for activation byimmobilized thrombin to convert fibrinogen to fibrin.

In a further optional embodiment, a needle designed to disrupt plateletsmay be used to apply a single-component concentrate (i.e., theconcentrated plasma) without the use of thrombin. FIG. 8 shows a syringe21 and applicator needle tip 23 separated by activator cartridge 22through which platelet-rich plasma passes for activation by, e.g., nylonwool. In this embodiment, calcium is added to the concentrateimmediately prior to or during its application to tissue in need ofsealing.

A porous membrane or other material with high exposed surface to voidvolume ratio can be derivitized with thrombin or other thrombogenic orthrombin-like enzyme by known methods. The derivatized membrane isfitted between syringe and applicator needle.

In a separate but related embodiment, glass wool, nylon wool or collagensponge or another material may be used in a similar configuration tocause eversion of the platelets, known to initiate coagulation.

In a separate but related embodiment, concentrated plasma may be keptcold to prevent gelation. After application, the heat of the livingtissue will initiate gelation.

A big problem with prior precipitation methods is that the fibrinogenmust be reconstituted. This is difficult and may take hours, and only arelatively low concentration is achievable. Thus not only does thepresent invention allow more rapid availability, a surgeon can decidethat "this one is a bleeder" and have sealant in 15 minutes. Previoustechnology requires that the surgeon order fibrin glue before theprocedure begins.

The present concentrated platelet-rich plasma can be left in the deviceuntil needed, for hours if necessary, without spontaneous gelation.

Other autologous precipitation methods require that blood be drawn daysin advance to allow time so that there is time for processing. Besidesthe inconvenience, this procedure allows the possibility of trackingerrors and thus infection.

Generally, a proteolysis inhibitor such as aprotinin is added to thefibrinogen component of fibrin glues. (Academic opinion is actuallydivided on whether this is necessary; need has never been provenrigorously.) The present methods for producing concentrated PRP retainthe natural inhibitors which are evidently lost in precipitation steps.

Fibrinogen from fresh plasma produces a mechanically superior gel (basedon the general properties of proteins damaged by precipitation).Platelets (contained in the present plasma concentrate) are useful forwound healing (many references available). Thus, an advantage of thepresent technology is that it provides better wound healing thanconventional fibrin glues, which do not contain platelets.

Surgeons, particularly in the U.S., are likely to use bovine thrombin to"activate" the present tissue sealant. This is not a regulatory problembecause bovine thrombin is available and is used routinely in surgery.However, the present invention provides a dispenser that activatesprothrombin present in the concentrated platelet-rich plasma and/or thatdisrupt platelets so that clotting is initiated by one or morecomponents in the concentrated plasma.

The present tissue sealant and autologous tissue sealant do more thanmerely seal the wound and adhere the damaged structures. In particular,the present autologous tissue sealant contains the patient's own livingthrombocytes and wound healing factors, and thus, it nurtures thehealing process. Other advantages of the present tissue sealant includethe presence of attachment factors which (i) improve adhesion to damagedtissue and (ii) promote cellular infiltration. In the present autologoustissue sealant, which is made from the patient's own blood, importantadvantages include the ease, speed and convenience of preparation (itmay be prepared in the operating room immediately before use).Furthermore, the optional absence of bovine thrombin in the presentautologous tissue sealant avoids immune system and foreign bodyreactions and eliminates disease transmission (i.e., from the bovinethrombin source).

I. Separator and Method for Separating Particulates from Suspensions

In a further aspect of the present invention, a separator and a generalmethod for separating particulates from suspensions is envisioned.Separating particulates from biological fluids (such as blood andplasma) is a common problem in the laboratory. A disposable cartridgecontaining a separation chamber (i.e., the first chamber in theapparatus used for preparing the present concentrated plasma) allowscellular and dense particle separation--basically anything that moves inresponse to centrifugal force.

Thus, the present apparatus for separating particulates from a liquidsuspension comprises:

an inlet,

a first chamber in fluid communication with the inlet, containing afirst separator for separating particulates from the liquid suspension,thus forming a particulate-free liquid, and

an outlet for withdrawing the liquid.

A "particulate-free liquid" is one which is substantially free ofinsoluble particles, preferably those insoluble particles having anaverage size above a pre-selected value (e.g., from 500 to 20 μ, or anyvalue for which filters or other devices are available for removing suchinsoluble particles; e.g., 250 μ, 100 μ, 80 μ, 50 μ, etc.).

J. Concentrator and Method for Concentrating Macromolecule Solutions

In an even further aspect of the present invention, a concentrator andmethod for concentrating macromolecule solutions based on the design ofthe concentrator (or second) chamber used in the apparatus for preparingthe present concentrated plasma is envisioned. SEPHADEX (trademark,Pharmacia), for example, is rarely used for concentrating macromoleculesolutions. The device may not concentrate to dryness, but is useful forconcentration of proteins (or any high molecular weight solution),preferably by a factor of three times or a multiple of three times,depending on the number of concentrator chambers in the apparatus.

Thus, the present apparatus for concentrating a solution of a substancehaving a molecular weight greater than a predetermined value (i.e.,macromolecule solution) comprises:

an inlet,

a chamber in fluid communication with the inlet, containing aconcentrator for concentrating the solution and a separator forseparating the solution from the concentrator, and

an outlet for withdrawing the concentrated solution.

Other features of the invention will become apparent in the course ofthe following descriptions of exemplary embodiments which are given forillustration of the invention and are not intended to be limitedthereof.

EXAMPLE 1

Using a cartridge as shown in either FIGS. 1, 2, 3 or 4, and followingthe procedure below, a concentrated platelet-rich plasma and tissuesealant can be prepared:

I. Collect blood from patient using standard citrate anticoagulant.

II. Inject a standard quantity of blood (e.g., 50 cc) in to the bloodfill port of the disposable cartridge. The cartridge is sealed makingcontamination impossible. It will be used in the operating room makingtracking errors impossible.

III. The operator then starts the microprocessor-controlled centrifugeby push-button. All remaining steps until removal of the concentrate areautomatic.

IV. (Optional step for designs incorporating free concentrator beads,FIG. 2) The centrifuge rotates briefly clockwise then counterclockwise afew times at a speed sufficient to evenly distribute the freedextranomer beads outside the concentrator cup lip.

V. The centrifuge spins at 3,000 rpm for one to ten minutes to separateplatelet-rich plasma from cells. The whole blood passes through a porein the blood receiver chamber into the separation chamber.

VI. The centrifuge stops for 15-90 seconds. Under the force of gravitythe platelet-rich plasma passes into the lower chamber, or concentratorchamber, (optionally by going through a through a hydrophilic filter,appropriate when free beads are present in the concentrator chamber asin FIG. 2).

VII. In the concentrator chamber platelet-rich plasma comes into contactwith beads bonded by electrostatic force to an open cell foam (cartridgeof FIGS. 1 and 3), free concentrator beads (cartridge of FIG. 2), orbeads embossed onto discs (cartridge of FIG. 4). Slow rotation of thecentrifuge aids mixing. The porosity and hydrophobicity of the porouswall are such that the low centrifugal force generated is not sufficientto overcome surface tension and the plasma remains within thecompartment at the inner surface. The beads swell with water, ions andlow molecular weight proteins (i.e., those proteins having a molecularweight below the cutoff value). The pore size of the beads is so chosenthat the proteins become concentrated in the space around the beads. Thequantity of beads is chosen so that the platelet-rich plasma isconcentrated the desired amount, e.g., 3×. When using DEBRISAN,approximately 2.6 gm is required per 10 cc plasma to achieve 3-foldconcentration. The protein concentrate contains the same ionconcentrations as the unconcentrated plasma.

VIII. The rate of rotation is increased forcing the concentratedplatelet-rich plasma out through the filter into the outer compartment.

IX. The centrifuge stops, and under the force of gravity concentratedplatelet-rich plasma collects in the bottom. The concentratedplatelet-rich plasma may be left for extended periods (hours) withoutspontaneous gelation.

X. Concentrated platelet-rich plasma is removed into a syringe.

XI. (Optional.) Thrombin/calcium solution is prepared separately.

XII. Using a variety of methods described in the literature forcryoprecipitated fibrinogen, the two solutions are combined and rapidlygel to form the TISSUE SEALANT.

XIII. (Optional; substitute for XI and XII.) Using a needle tip designedto disrupt platelets, a single-component concentrate may be appliedwithout the use of bovine thrombin.

A comparison of the properties of the present tissue sealant and priortissue sealant prepared from cryoprecipitated fibrin is shown in theTable below:

                  TABLE                                                           ______________________________________                                        Concentrations of Tissue Sealant Compositions                                 Blood     Precipitate Fibrin                                                                           Autologous                                           Component Glue           Concentrated PRP (3X)                                ______________________________________                                        Ions      per reconstitution                                                                           identical to patient                                           solution       plasma                                               Platelets none           3X plasma                                                                     concentration                                        Proteins  only proteins that                                                                           all plasma proteins                                            cryoprecipitate;                                                                             at normal (less than                                           fibrinogen, factor                                                                           5 kDa) or 3X normal                                            XIII, fibronectin,                                                                           (more than 5 kDa)                                              albumin, plasminogen                                                                         concentrations                                       Albumin   10-25 mg/ml    100-165 mg/ml                                        Factor XIII                                                                             75 μg/ml    30-60 μg/ml                                       Fibrinogen                                                                              70-110 mg/ml   5-14 mg/ml                                           Fibronectin                                                                             2-9 mg/ml      0.5-1.2 mg/ml                                        Plasminogen                                                                             20-60 μg/ml 600 μg/ml                                         Aprotinin 1,500 KIU/ml   0 KIU/ml                                             ______________________________________                                    

The present tissue sealant also appears to show similar tensile strengthto that of cryoprecipitated fibrin tissue sealant. The adhesive strengthprovided by the present tissue sealant is believed to be superior tothat of cryoprecipitated fibrin tissue sealant because fibrinogen andother important proteins are not denatured by the present process.

A key advantage of the present invention is the presence of platelets.Adhesion of the platelets to collagen III fibrils leads to plateletaggregation, promoted by 5-hydroxytryptamine and epinephrine releasedfrom the platelets. Other growth and healing factors are released by theplatelets. Platelet-derived growth factor is a mitogen for fibroblastsand smooth muscle cells. Platelet factors stimulate neovascularization,especially when the area is anoxic. Platelets also make fibrin moreresistant to mechanical shear forces and to fibrinolysis.

It is possible that for certain applications lesser numbers of plateletsmay be desirable. The invention may be adjusted to achieve this outcome.Because of their low mass compared with other blood cells, apreponderance of platelets is preserved in the final products whenconditions are chosen to minimally separate the bulk of said moremassive cells from whole blood. For example, a 4-inch diametercartridge, 1 inch in height and fitted with a sintered plastic wall asin FIG. 2 will separate most of the blood cells from 50 cc of wholeblood when centrifuged for 3 minutes at 2,000 rpm. By increasing thespeed to 5,000 rpm and the time to 15 minutes, most of the platelets arealso sedimented through the porous walls to yield plasma depleted ofplatelets. Such platelet depleted plasma may be desirable if, forexample, it were desired that the plasma or concentrate derivedtherefrom should be processed by filtration through a sterile submicronmembrane to render the final product suitable for storage for anextended time (days).

Obviously, numerous modifications and variations of the presentinvention are possible in light of the above teachings. It is thereforeto be understood that within the scope of the appended claims, theinvention may be practiced otherwise than as specifically describedherein.

45. See, for example, DelRossi, A. J., A. C. Cernaianu, R. A. Vertrees,C. J. Wacker, S. J. Fuller, J. Cilley Jr., and W. A. Baldino."Platelet-rich plasma reduces postoperative blood loss aftercardiopulmonary bypass." J Thorac Cardiovasc Surg 100 (2 1990): 281-6.

What is claimed as new and is desired to be secured by letters patent ofthe United States is:
 1. A method of making concentrated plasma for useas a tissue sealant or adhesive, comprising the steps of:(a) separatingplasma from whole blood; (b) removing water from said plasma bycontacting said plasma with a concentrator which does not significantlydenature fibrinogen to provide said concentrated plasma; and (c)separating said concentrated plasma from said concentrator.
 2. Themethod of claim 1, wherein said separating step (a) comprisescentrifugally contacting said whole blood with a separator which barspassage of red blood cells and white blood cells in the absence of anapplied centrifugal force, and applying a centrifugal force effective topass red blood cells and white blood cells in said whole blood throughsaid separator.
 3. The method of claim 2, wherein said separator is amat, foam or porous plastic annulus.
 4. The method of claim 1, whereinsaid separating step (a) comprises centrifugally settling said wholeblood into a chamber shaped and positioned such that gravity does notpermit drainage of said red blood cells and said white blood cells inthe absence of an applied centrifugal force.
 5. The method of claim 1,wherein each of said whole blood and said plasma contains (i) platelets,(ii) high molecular weight compounds or (iii) both platelets and highmolecular weight compounds.
 6. A platelet-rich plasma concentrate,prepared by the process of claim
 5. 7. The method of claim 1, whereinsaid concentrator comprises a material selected from the groupconsisting of a dextranomer, dextramine, polyacrylamide, silica gel, azeolite, cross-linked agarose, starch and alginate gel, either in freebead form or attached to a surface.
 8. The method of claim 7, whereinsaid material is attached to a matrix, and said matrix comprises a mat,a foam or a disc.
 9. The method of claim 1, wherein said blood containsan amount of an added coagulation inhibitor which is effective toinhibit coagulation of said blood.
 10. The method of claim 1, conductedin a sealed cartridge having an inlet, a first chamber containing afirst separator for separating said plasma from said whole blood, asecond chamber containing said concentrator and a second separator forseparating said concentrated plasma from said concentrator, and anoutlet for withdrawing said concentrated plasma.
 11. The method of claim1, further comprising, after said separating step (c), repeating saidcontacting step (b) and said separating step (c).
 12. A plasmaconcentrate, prepared by the process of claim
 1. 13. The plasmaconcentrate of claim 12, wherein said plasma concentrate is autologous.14. A tissue sealant, comprising the plasma concentrate of claim 12 andan activator.
 15. The tissue sealant of claim 14, wherein said activatoris a mixture of thrombin and calcium in amounts sufficient to permitgelation of said platelet-rich plasma concentrate.
 16. The tissuesealant of claim 14, wherein said activator is autologous thrombin andplatelets.
 17. A method of sealing tissue, comprising the steps of:(a)preparing a plasma concentrate according to the process of claim 1, (b)activating said plasma concentrate to provide an activated plasmaconcentrate, and (c) applying said activated plasma concentrate totissue in need of sealing.
 18. The method of claim 17, wherein saidplasma concentrate is obtained from blood drawn from a patient, and saidtissue in need of sealing is in or on said patient.
 19. The method ofclaim 17, wherein said activating comprises contacting said plasmaconcentrate with a mixture of thrombin and calcium in amounts sufficientto gel said activated plasma concentrate.
 20. A method of sealingtissue, comprising the steps of:(a) preparing a plasma concentrateaccording to the process of claim 1, said plasma concentrate containingprothrombin, platelets or both prothrombin and platelets, (b) activatingsaid prothrombin, said platelets or both said prothrombin and saidplatelets to provide an activated plasma concentrate, and (c) applyingsaid activated plasma concentrate to tissue in need of sealing.
 21. Themethod of claim 20, wherein said activating comprises irradiating saidplatelets with ultrasound sufficiently to result in gelation of saidactivated plasma concentrate.
 22. The method of claim 20, wherein saidactivating comprises heating said plasma concentrate sufficiently toresult in gelation of said activated plasma concentrate.
 23. Theapparatus of claim 21, wherein said concentrator has a molecular weightcutoff of from 2 to 100 kDa.
 24. The apparatus of claim 21, wherein saidconcentrator is selected from the group consisting of a dextranomer,dextramine, polyacrylamide, silica gel, a zeolite, cross-linked agarose,starch and alginate gel, either in free bead form or attached to asurface.
 25. The apparatus of claim 21, wherein said concentrator isembedded in or attached to a surface.
 26. The apparatus of claim 25,wherein said matrix comprises a plurality of discs.
 27. A plasmaconcentrate comprising platelets, from 5 to 400 mg/ml of fibrinogen,from 0.5 to 35 mg/ml of fibronectin, and a physiologically acceptablecarrier comprising water and physiologically acceptable inorganic andorganic ions in a physiological acceptable concentration, wherein thefibrinogen in the concentrate is not significantly denatured.
 28. Theplasma concentrate of claim 27, prepared from whole blood, wherein saidplatelets are present in said plasma concentrate in a concentration ofat least one-and-one-half times the concentration of said platelets inunconcentrated plasma from said whole blood.
 29. The plasma concentrateof claim 27, further containing a compound selected from the groupconsisting of an antibiotic, a collagen fleece, collagenase, hyaluronicacid, a wound-healing factor, zinc ions in an amount effective todecrease coagulation time of said plasma, and a biologically acceptabledye.
 30. The plasma concentrate of claim 29, further containingdisulphine blue in an amount sufficient to visibly detect said plasmaconcentrate during its application to tissue.
 31. The plasma concentrateof claim 27, further comprising a pharmaceutically active compound. 32.A method of making concentrated plasma for use as a tissue sealant oradhesive, comprising the steps of:(a) separating plasma from wholeblood; and (b) removing water from said plasma with a concentrator whichdoes not significantly denature fibrinogen to provide said concentratedplasma.
 33. A method of claim 32 wherein the concentrator is awater-absorbent.
 34. A method of claim 33 wherein the concentrator is amaterial selected from the group consisting of a dextranomer dextramine,polyacrylamide, silica gel, a zeolite, cross-linked agarose, starch andalginate gel.
 35. A method of claims 34 wherein the concentrator is afree bead form or attached to a surface.
 36. A method of claim 32wherein the concentrator includes a membrane.
 37. An apparatus with adisposable cartridge for concentrating plasma for use as a tissuesealant or adhesive, comprising a centrifuge separator means forseparating plasma from whole blood; and concentrator means in fluidcommunication with said centrifuge separator means for removing waterfrom said plasma without significantly denaturing fibrinogen, to provideconcentrated plasma.
 38. An apparatus of claim 37 comprising a firstchamber containing the centrifuge separator for separating plasma fromwhole blood; and a second chamber in fluid communication with said firstchamber, the second chamber including a concentrator means forconcentrating said plasma by removing water from said plasma withoutsignificantly denaturing fibrinogen and a second separator forseparating concentrated plasma from said concentrator.
 39. An apparatusof claim 38 wherein the concentrator means comprises a material selectedfrom the group consisting of a dextranomer, dextramine, polyacrylamide,silica gel, a zeolite, cross-linked agarose, starch and alginate gel.40. An apparatus of claim 39 wherein the concentrator means is a freebead form or attached to a surface.
 41. An apparatus of claim 38 whereinthe concentrator means includes a membrane.
 42. A plasma concentratecontaining fibrinogen for use as a tissue sealant or adhesive, thefibrinogen in said concentrate not being significantly denatured.
 43. Aplasma concentrate of claim 42 containing from 5 to 400 mg/ml offibrinogen.
 44. A plasma concentrate made by the process of claim 38.45. An apparatus with a disposable cartridge for concentrating plasmafor use as a tissue sealant or adhesive, comprising:an inlet, a firstchamber in fluid communication with said inlet, the first chambercontaining a centrifuge separator for separating plasma from wholeblood, a second chamber in fluid communication with said first chamber,the second chamber containing a concentrator for concentrating saidplasma by removing water from said plasma without significantlydenaturing fibrinogen and a second separator for separating concentratedplasma from said concentrator, and an outlet for withdrawing saidconcentrated plasma.